Hi all,
Here's the situation: We have used a 30% PEG8000 and 20% PEG400 mix to
stabilize our crystals and to act as cryoprotectant. We soaked in a
pentasaccharide substrate into the crystal (or at least we added the substrate
to the soak solution and allowed the crystal to soak several hours) and
collected data at -100C.
The maps do show partial ring structures in the binding site, but the occupancy
is very low and there is no connectivity. In the spaces between protein
molecules, we see long stretches of connectivity.
Questions:
1) Is the long connectivity PEG400?
2) Since PEG400 is at 20%, can PEG400 prevent the saccharide substrate from
soaking into the crystal since the percentage of saccharide is much lower than
the percentage of PEG400?
3) Has anyone had similar experiences seeing PEG400 in their maps? If so, do
you have a structure/parameter/tophology of PEG400?
4) Can we use the substrate itself as a stabilizer and cryoprotectant to
replace PEG400? Say use a polymer of the substrate at about the same molecular
weight and at the same percentage?
5) Other than sucrose, is there any other disaccharide which has been shown to
be a good cryoprotectant?
Any and all help, tricks, comments (good or bad) and suggestions are welcomed.
--
Robert D Scavetta
Dr. Frances Jurnak's Laboratory
Department of Biochemistry
1447 Boyce Hall
University of California
Riverside, CA 92521
email: scavetta at xtreme.ucr.edu
phone: (909) 787-4196
fax: (909) 787-3590